Process for the oxygenation of steroids with nigrospora



United States Patent-O PROCESS FOR OXYGENATION F STEROID WITH NIGROSPORA Robert D. -'-Muir, Deerfiel'd, and Raymond M. Dodson, 1 1

Park Ridg'e lll -assignors to G.D.:Searle& (30., Chicago-,1'Ill., a-corporation of Delaware- This-invention relates to a novel method 'fbr the introduction' of oxyg'en into a steroid m'ol'eculeyand is particularly concerned "with the process" of oxygenatirlg' ice 2 able to its'propagation. 'Mo'istcereal grits, illustratively hominy grits, are suitablemedia' for the propagation of Nigrospora in the laboratory. However, for the process of the present invention, synthetic or partiallyv synthetic liquid media are generally preferred", as .such liquidv media afford 'c'ertainiadvautages: Incorporation of availablecarbon'; nitrogen, rhitieralsandlaccessory nutrient factors is faeilitatedre the use of a 1iquid.n'1'edium.- .Use of a liq'u nltn'fmed'ium afiords additional advantage in permitting etfi ihtaeratit'iii, intimate contact of the steroid suhs'tralte 'v'vith the joxy'genating enzymes of the fungus and eonveniences:inithe isolation of the. final product; Such a cul n diam can incorporate-a carbohydrate as a source; of avail'ahle carbon, and proteins, hydroly is products of pioteir 'js', or inorganic nitrates or ammonium sans-as a senrceef availahle nitregen. Additionaltii'u-l t'rier'l .raefers can" he supp ied by the incorpo ation of corn steep liqiior inttithe' rriedium. Miheral constituent-s. other accessory growth-factors recogniied in the pilot art as Being necessary or desirable for. th'e'growth of fungi are" nsna1'1y"natura11y present in the'culture-media' properties, but have not heretofore been c'onveni'eiitly or economically availablefrom known synthetic orbio-2 chemical processes. The process of the present inven tion provides an excellent route-tooxygenated steroids,- including l5-oxygenated steroids, and constitutes an im'-" portant advance over the'prio'r art.-

The organisms employed in the process. ofthefpresetit invention are oxygenating strains of fungiof the genus Nigrospora, which genus belongs. to. the order .Moniliales of the class'Fungilmperfecti; Comprehended w ithip th'e genus Nigrospora are various strains which have. some* times, especially in the earlier literature; been"assigried' different names; The classification of E. W. Mason; Transactions of the ennsh-Myeelegiear Society, "1 2, 152-165 (1927) proposes theacceptance of three species In this classification strains included" withinN. ioryz'ae are-Monotospora 0ryza'e,--Mem'n0izillm= pal-micolum, Tfi chosporiilm* palmic'olum, Acremoniella= 'bcculta, Basis= porium gallarum; 'Glen0spora elasticate; and possibly Sporotrichum ma ydis, Tric'hosporium maydis, and My cogone nigra. Strains included within N. sphae rica are Trichoisporium s'phae'ricum, 'Hadrotrichum' aru'ndir'iac'eum, Epicbccum le visporum, Epicoccum hyalopes, Conicsporium g'ecev-i; 'Coniosporium extremorum, and possibly M'ono'tospora higr'a. N. sacchari is used synonymously with-' 'the earlier designation Glenospora sacchari. Other strains of organisms of the genus Nigrospora have appeared under the names N. panici, N. i'avanica andpossibly Coniospo'rium micans.

Cultures of organisms of the genus Nigrospora, suit able: for carrying out the process of this-invention, can be obtained-frompublic culture collections, Alterna tivel-y; organisms of this fungal genus can be isolated from natural sources by standard procedures and tech niques well known to microbiologists. Nigrosporafung'i described hereinafter. Whe1i-synthetic mediaearener'nploy'ed which. are deficient in mineralsv and other. accessory growth factors, such constituents areaddedlas re q e 1 Thel sterilized culture medium is inoculated .with .the selected species of Nigros'porafung'us andgrowth of the fungus is allowed. to proceed .uriderxaerohicl conditions at a suitable temperature. 1 Temperatures. within the range of 20250 C. have. been founddesirable for the. propaga; tion I of l Ni'grospora .fufngi' although 1 appreciable. growth also? takes. place outside of: .these'. lir'iiits.v .wlien euuure media are usedwhich are primarilysolid anclpar ticulate, such as. moist cereal; grits, satistactory' aeratiomis. achieved by such meansas growing the. fungusin relatively. flat layers or trays or bygrowingitin a rotating drum; .Aeration of liquid culture media-is conveniently;accomplished by agitationsuchas is achivedbystirringor ,byuse of a shaking. platform, by blowing...air' through. the culture medium,.or by a corn binationnof v .these:means.. Therexact time andmanneriof ntroducin the steroidal substrate. to. the orygenatingwactivityaofathe .Nigrospora fungus. is a relativel[non-criticalfactor and-these. operatiflgdetails can be selectedfrorrr .Withinl. fair-1y; wide limits. For example, theasteroid can. be: added. to 1 the culture medium prior to sterilization,atthe-time-of'inocu lation of the medium with the fungus, or after the fungus has been allowed to grow "for" aperiod of time, conveniently up to 48 hours. :The: :steroidito be :oxygena-ted can be added'to the-culture medinmzastasolid, a; suspe'ne sion; or a so'lutionr A desirably finezsta-te of:dispersion:of the steroid is achieved;- bysaddingeit to the": feniientatioh medium: as: a 'solutiom in a small .a'mount 30f ethanol. Oxygenation ofi-the steroid-:can-be achieved: by subjecting it to theactionof the'eiiti'r'e cul'turei'm'e'tiiumporaltei'e natively, with the useaiofap'r'ocedtlres rknown to: thezprior art-,by subjecting it 't'o, the action: of-sthe separated. OX): genatirfg enzymes, free); of nutritional" factors a whichaare needed fo'r the growthofi the fungus: r t t 1 Satisfactory yields ofoxygenatedpfr'odiictsarei-obtained by. allowing a: contact.tirne? of, typically; 24'orw48 hours; althoughsubstantia]; conversion beginsahnost immediately after the addition of thesteroid' to the fermenta'tion' mixeture. fThe-minimumperiodof time'req'uired for; aalsatis factorwdegfeet of conversion of vthei steroid depends on various factors such as -th'e" 'stageof: growth of. the. fungus at the time of the addition; of thersteroid,theftemperature' offerrnentaitorr and the "rate" of aeration Intgener-al; extensivetgrowth? of the; fungus before the. addition: of the steroid substrate tends to dessert the: contact timei required for satisfactoryoxygnatioii of the steroid. period-of i fungal growth 'of 48:11am, followed b'y a steroid easiest 3. time of 24 hours, has been found to give generally good results when the process of the present invention is carried out as described in the examples given hereinafter.

At the conclusion of the fermentation period, the result? ing steroid or mixture of steroids is recovered from the fermentation mixture. Depending on the contact time and other operating factors, the recovered steroidal product may contain unreacted starting material as well as oxygenated steroid The crude steroidal product can be recovered by extracting the fermentation mixture with a water-immiscible solvent. Water-immiscible ethers, esters and chlorinated hydrocarbons are especially suitable for this purpose. It is convenient to separate the mycelium from the fermentation liquor by filtration prior to extraction with the organic solvent, as emulsions are minimized and the mycelium and liquid phase are more easily processed when they are treated separately. The mycelium and filtrate are both extracted with a suitable solvent and the combined extract, which can be washed with dilute aqueous sodium bicarbonate solution and with water, is concentrated to dryness, affording a crude steroidal product which usually contains other components such as nitrogenous materials. Purification by chromatographic fractionation or by recrystallization affords the desired oxygenation products.

Steroidal compounds which are oxygenated in the 15 position are physiologically active and are useful in chemotherapeutics. They have hormonal properties and are useful in the treatment of states of hormonal deficiencies. The compounds described herein are valuble hypotensive agents; for example, both the 15-hydroxyprogesterone and the dihydroxyprogesterone resulting, as described hereinafter, from the fermentation of progesterone with Nigrospora produce a fall in blood pressure following their administration. Since Nigrospora fungi are capable of oxygenation at position 11 as well as at position 15, as disclosed hereinafter, the process of the present invention is also useful for the production of intermediates of value in the synthesis of adrenocortical hormones.

This invention will appear more fully from the examples which follow. These examples are set forth by way of illustration only and it will be understood that the invention is not to be construed as limited in spirit or in scope by the details contained therein. It will be apparent to those skilled in the art that many modifications in materials and methods can be made without departing from the scope of this invention. In these examples, temperatures are given in degrees centigrade (C.). Except where otherwise indicated, quantities of materials are given as parts by weight.

EXAMPLE 1 A nutrient medium is prepared, having a composition of 28 grams of a commercial enzymatic digest of lactalbumin, 50 grams of dextrose and 3 grams of corn steep liquor, diluted to 1 liter with tap water and adjusted to a pH of about 4.5 with concentrated hydrochloric acid. A total of 8 liters of this medium is dispensed in equal portions of 400 milliliters into 20 Erlenmeyer flasks having a capacity of 2 liters each. The flasks are plugged with non-absorbent cotton, and are sterilized by beating them it: an autoclave for minutes at about 120 C. The flasks are cooled and the contents of each are inoculated with a culture of Nigrospora oryzae, American Type Culture Collection No. 8667. They are then mounted on a shaking platform which operates in a manner such that every point on the platform describes a circle of about 2% inches in diameter at a rate of approximately 225 revolutions per minute. conditions are recommended, it has been found that within fairly wide limits the rate of aeration of the culture media is not a critical factor, and satisfactory results are obtained with other rates of agitation or by other means of aeration, such as by the introduction of sterilized air or oxygen through an inlet tube. Growth of the organism is allowed to proceed for about 48 hours. To each ofthe While these specific operating flasks there is then added a solution of 100 milligrams of progesterone in 3 milliliters of ethanol. Incubation is continued on the shaking platform at approximately 25 C. for 26 hours after the addition of the steroid substrate. After this period of fermentative oxygenation of the steroid, the contents of the 20 flasks are combined and filtered in order to separate the mycelium from the liquid phase. The mycelium is washed with 2.8 liters of methylene chloride, and this wash liquor is added to fresh methylene chloride which is used to extract the aqueous filtrate. The aqueous filtrate is extracted with. a total of 11.2 liters of methylene chloride in 2 portions. The combined methylene chloride extracts are concentrated to dryness under reduced pressure, affording about 4.2 grams of a non-crystalline residue. This material is dissolved in a 10 volume percent solution of ethyl acetate in benzene and fractionated on a chromatography column prepared from 420 grams of silica. The column is developed'with 1 liter portions of solvents. Results of a representative fractionation are given in Table I and in the text which follows. It will be obvious to anyone possessing ordinary skill in chromatographic separations that the absolute positions of elution from the column are subject to variations in different runs, and that residues from successive eluates are combined for purification on the basis of evidence of substantial homogeneity, such as is afforded by a showing that successive residues have identical or similar melting points.

Table I Eluate Solids,

Fractions Milligrams Ethyl Acetate.--

The crystalline residues from fractions 23 through 25, washed with ether, melt in the range of 129-166 C. Purification by recrystallization from a mixture of acetone and cyclohexane, then from dilute acetone, and finally again from a mixture of acetone and cyclohexane affords a compound melting at about 187188 C. and exhibiting a specific rotation of about +1765 in chloroform solution. The crystalline residues from fractions 28 through 32, Washed with ether, melt in the range of 2l5-229 C. They are combined and recrystallized from acetone and then from dilute methanol to yield 15a-hydroxy-4- pregnene-3,20-dione (l5a-hydroxyprogesterone) melting at 228-230 C. This preparation exhibits a specific rotation of +226 in chloroform solution and an ultraviolet absorption maximum at 241 millimicrons with a molecular extinction coeflicient of 18,200. The crystalline residues from fractions 34 through 38, washed with ether, melt in the range of -164 C. and consist chiefly of a diketopiperazine. The crystalline residues from fractions 49 and 50, washed with ether, melt in the range of ISO- C. They are combined and recrystallized twice from a mixture of acetone and cyclohexane to yield 11a,1SB-dihydroxy-4-pregnene-3,ZO-dione (l1or,15 3-dihydroxyprogesterone) melting at 202-203 C. This compound has a specific rotation of +l34 in chloroform solution and an ultraviolet absorption maximum at 240 millimicrons' with a molecular extinction coefiicient of 17,100. Its ultraviolet absorption spectrum does not change appreciably with time in aqueous potassium hydroxide. I

EXAMPLE 2' A solution of 1 part of 1 111,1S S-dihydroXyA-pregnene- 3,20-dione in a mixture of 40 parts of pyridine and 40 parts of acetic anhydride is allowed to stand at room temperature for about 16 hours. The reactionmixture is diluted with water to several times its original volume and extracted with methylene chloride. The organic phase is washed with dilute hydrochloric acid and with water, and is then dried over sodium carbonate, filtered and concentrated to dryness. The residue in benzene solution is poured on a chromatography column prepared from 60 parts of silica. The column is eluted with mixtures of ethyl acetate and benzene containing increasingly higher percentages of ethyl acetate. Elution with a 30 volume percent solution of ethyl acetate in benzene affords 1101,1SB-diacetoxy-4-pregnene-3,20-dione melting at 179- 181 C. This compound has a specific rotation of +136 in chloroform solution and an ultraviolet absorption maximum at 239 millimicrons with a molecular extinction coefficient of 17,200.

EXAMPLE 3 A solution prepared from 1 part of 11u,15fi-dihydroxy- 4-pregnene-3,20-dione, 5 parts of pyridine and 5 parts of acetic anhydride is allowed to stand at room temperature for 30 minutes. The reaction mixture is diluted to several times its original volume with ice water, and the precipitated product is collected on a filter and washed. Recrystallization from a mixture of acetone and cyclohexane yields 11a acetoxy 15 8 hydroxy-4-pregnene-3,20-dione melting at 233-235 C. This compound has a specific rotation of +l1l;6 in chloroform solution and an ultraviolet absorption maximum at 239.5 millimicrons with a molecular extinction coeflicient of 17,000.

EXAMPLE 4 A solution of 1 part of 11a,15B dihydroxy-4-pregnene- 3,20-dione in 40 parts of pyridine is stirred with amixture of 2 parts of chromium trioxide in 80 parts of pyridine and allowed to stand at room temperature for about 16 hours. The reaction mixture is poured into several'time's its volume of water and extracted with 3 portions of methylene chloride. The methylene chloride phase is washed with water, dried over sodium sulfate, filtered and concentrated to dryness. The residue in benzene solution is poured on a chromatography column prepared with 60 parts of silica, and the column is eluted withmixtures of benzene and ethyl acetate containing gradually increasing amounts of ethyl acetate. Elution with a 30 volume percent solution of ethyl acetate in benzene, followed by concentration of the eluates and recrystallization of the residues from a mixture of acetone and cyclohexane and finally from aqueous acetone, yields 4-pregnene-3,11,15,20- tetrone (11,15 dioxoprogesterone) melting at 218.5- 221.5 C. This compound has an ultraviolet absorption maximum at 238.5 millimicrons with a molecular extinction coefficient-of 17,700. The infrared absorption spectrum includes a prominent band at 5.75 microns indicative of a 5-membered ring ketone. Other principal infrared absorption maxima appear at 5.89, 5.97, 6.19, and 7.36

microns.

EXAMPLE 5 A solution of 5 parts of 1111,15B-dihydroxy-4-pregnene- 3,20-dione in 300 parts of glacial acetic acid is mixed with a solution of 2 parts of chromium trioxide in 100 parts of water and allowed to stand at room temperature for 1 hour. The mixture is diluted with several times its volume of water and extracted with methylene chloride. The organic phase is concentrated to dryness, and the residue in benzene solution is poured on a chromatography column prepared from 300 parts of silica. The

column is eluted with mixtures of benzenerand ethyl acetate containing gradually increasing proportions of ethyl acetate. Elution with a. 30 volume percent solution of ethyl acetate in benzene affords -4pregnene-3,11, 15,20-tetrone, identical with the product of Example 4. Elution with a 40 volume percent .solution of ethyl acetate in benzene, followed by concentration .of the eluates and recrystallization of the residues from a mixture of acetone and cyclohexane yields 11u-hydoxy-4-pregnene-3,15,20- trione having a melting point of 187-189 C., and an ultraviolet absorption maximum at 240 millimicrons with a molecular extinction coefficientbf 17,700. Prominent infrared absorption maxima appear at 2.94, 5.76, 5.92, -6;04, 6.24, and 7.36 microns. Elution with a 70 volume percent solution of ethyl acetate in'benzene removes a small amount of "unreacted 11ml5B dihydroxy-4 pregnene-3,20-dione from the column.

EXAMPLE v6 By the procedure of Example 1, progesterone is fermented with a 48 hour growth 0f N'igrospara sp., ATCC 12773. The oxygenation products are recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 7 By the procedure of Example 1, progesterone is fermented with a 48 hour growth of Nigrospora sphaerica, ATCC 12772. The oxygenation products are obtained by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 8 By 'the procedure of Example 1 a 48 hour growth of Nigrospora oryzae ATCC. 12771, is prepared. To each of the 20 culture flasks there is then added a solution of milligrams of 17a-hydroxy-4-pregnene-3,20+dione (17-hydroxyprogesterone) in 5 milliliters of ethanol. Incubation is continued on the shaking platform at approximately 25 C. for 72 hours after the addition of the steroid substrate. After this period of fermentative oxygenation of the steroid, the contents of the 20 flasks are combined and filtered in orderto separate the mycelium from the liquid phase. The mycelium and the filtrate are extracted with methylene chloride, and the combined methylene chloride extracts are concentrated to dryness under reduced pressure, affording about 2.7 grams of a non-crystalline residue. This residue is dissolved in a 5 volume percent solution of ethyl acetate in benzene and fractionated on a chromatography column prepared from 200 grams of silica. The column is developed with 500 milliliter portions of solvents. Results of a representative fractionation are given in Table II and in the text which follows.

The crystalline residues from fractions 22 through 24, washed with ether; melt in the range of 200-215 C. Purification by recrystallization from mixtures'of acetone and cyclohexane yields 1513,17e-dihydroxy-4pregnene- A solution of parts of 1513,17a-dihydroxy-4-pregnene- 3,20-dione in 300 parts of pyridine is'added to a suspension of 5 parts of chromium trioxide in 400 parts of pyridine and allowed to stand at room temperature for 24 hours. The reaction mixture is diluted with several times its volume of water and extracted with 3 portions of a mixture of equal parts of benzene and ether. The organic phase is separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. Purification of the residue by recrystallization from a mixture of acetone and cyclohexane and finally from dilute acetone yields 17,8-hydroxy-4-pregnene-3,15,20-trione melting at 218-220 C. and having a specific rotation of +133 in chloroform solution. This compound has an ultraviolet absorption maximum at 238.5 millimicrons with a molecular extinction coefiicient of 17,500.

EXAMPLE A total of 8.8 liters of the nutrient medium described in Example 1 is dispensed in equal portions of 400 milliliters into 22 Erlenmeyer flasks having a capacity of 2 liters each. The contents of the flasks are sterilized, cooled and inoculated with a culture of Nigrospora oi'yzne, ATCC 12771. By the procedure described in Example 1, a 24 hour growth of this organism is obtained. To each of the flasks there is then added a solution of 100 milligrams of 21-acetoxy-4-pregnene-3,20-dione (desoxycorticosterone acetate) in 5 milliliters of ethanol and incubation is continued on the shaking platform at approximately 25 C. for 96 hours after the addition of the steroid substrate. The contents of the 22 flasks are combined and filtered in order to separate the mycelium from the liquid phase, and both are extracted with methylene chloride. The methylene chloride extracts are concentrated to dryness under reduced pressure, affording about 4.1 grams of a non-crystalline residue. This material is dissolved in a volume percent solution of ethyl acetate in benzene and fractionated on a chromatography column prepared from 350 grams of silica. The column is developed with 1 liter portions of solvents. Results of a representative fractionation are given in Table III and in the text which follows.

Table III Solvent, Volume Percent Ethyl Acetate in Benzene Eluate Solids,

Fractions Milligrams tallization from acetone affords a monohydroxy substitution product of 21-hydroxy-4-pregnene-3,ZO-dione melting at 210213 C. and having a specific rotation of +214 in chloroform solution. This compound has an ultraviolet absorption maximum at 241.5 millimicrons with a molecular extinction coefiicient of 17,100. It is l5cc,2l dihydroxy-4-pregnene-3,ZO-dione, derived from desoxycorticosterone acetate by hydrolysis of the ester grouping and monohydroxylation at position 15.

EXAMPLE 11 A culture of Nigrospora sp., AT CC 12774, is grown for 66 hours in 20 flasks, according to the procedure of Example 1. To each of the flasks there is then added a solution of 100 milligrams of progesterone in 3 milliliters of cthanol and incubation is continued on the shaking platform at approximately 25 C. for 22 hours after the addition of the steroid substrate. The contents of the 20 flasks are then combined and filtered in order to separate the mycelium from the liquid phase. The mycelium and the filtrate are extracted with methylene chloride and the combined methylene chloride extracts are concentrated to dryness under reduced pressure, affording about 4.2 grams of a non-crystalline residue. This material is dis solved in a 5 volume percent solution of ethyl acetate in benzene and fractionated on a chromatography column prepared from 400 grams of silica. The column is developed with 1 liter portions of solvents. Results of a representative fractionation are given in Table 1V and in the text which follows.

Table IV Eluate Solids,

Fractions Milligrams Ethyl Acetate..-

The crystalline residues from fractions 24 through 31, washed with ether, melt in the range of 208 C. Combination of these residues and purification by recrystallization from acetone and from dilute methanol yield 15u-hydroxy-4-pregnene 3,20-dione, identical with the product of Example 1. The crystalline residues from fractions 34 through 36 consist principally of a diketopiperazine.

EXAMPLE 12 A solution of 54 parts of 15a-hydroxy-4-pregnene-3,20- dione in 3000 parts of pyridine is added to 58 parts of chromium trioxide in 4000 parts of pyridine and the mixture is allowed to stand at room temperature for 18 hours. The mixture is diluted with several times its volume of water and extracted with 3 portions of a solvent mixture containing equal parts of benzene and ether. The combined organic extract, is washed with water and concentrated to dryness in a vacuum. Recrystallization of the residue from dilute methanol and then from a mixture of acetone and petroleum ether affords 4-pregnene-3,l5, 20-trione melting at 154455 C. and having a specific rotation of +2186 in chloroform solution. The infrared absorption spectrum shows a prominent band at about 5.76 microns, indicative of a 5-membered ring ket ns.

9 EXAMPLE 13 In the same manner as Example 1, testosterone is fermented with a culture of Nigrospora oryzae, ATCC 8667, and the resulting steroidal product is recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 14 In the same manner as Example 1, 4androstene-3,17- dione is fermented with a culture of Nigrospora oryzae, ATCC 8667, and the resulting steroidal product is recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 15 In the same manner as Example 1, 17u,21-dihydroxy- 4-pregnene-3,20-dione is fermented with a culture of Nigrospora oryzae, ATCC 8667, and the resulting steroidal product is recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 16 In the same manner as Example 1, 2l-hydroxy-4- pregnene-3,20-dione (desoxycorticosterone) is fermented with a culture of Nigrospora oryzae, ATCC 8667, and the resulting steroidal product is recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 17 In the same manner as Example 1, 16,17-epoxy-4- pregnene-3,20-dione is fermented with a culture of Nigrospora oryzae, ATCC 8667, and the resulting steroidal product is recovered by extraction with methylene chloride and chromatographic fractionation on silica gel.

EXAMPLE 18 A stainless steel fermentation tank having a capacity of about 40 liters is charged with a solution of 1000 grams of dextrose, 200 grams of a commercial enzymatic digest of lactalbumin, 90 milliliters of corn steep liquor and 15 milliliters of concentrated hydrochloric acid in liters of tap water. Five grams of an antifoaming agent, suitably of a silicone-type, is added and the contents of the vessel are sterilized by the introduction of live steam under pressure to a final temperature of 110 C. and a final volume of about liters. The contents of the fermentor are cooled and inoculated with a culture of Nigrospora oryzae, ATCC 8667. The contents of the fermentor are kept agitated by a stirrer operating at about 200 revolutions per minute. A stream of air which has been sterilized by filtration through a glass wool filter is introduced through an inlet tube into the fermentor at a rate of from 15 to 22 liters of air per minute. This rate of aeration is measured by means of a rotameter placed in the sterile portion of the air line. Growth of the organism is allowed to continue for 48 hours at 25 C., during which time the pH of the fermentation medium drops from about 4.4 to 3.7. During the 48 hour period of growth of the organism, two additional portions of 5 grams each of an antiforming agent are added. A solution of 7.5 grams of progesterone in 500 milliliters of ethanol is added and fermentation in the presence of the steroid substrate is continued for an additional 24 hours at 25 C., with the same rates of stirring and aeration. The contents of the fermentor are stirred for one hour with 18 liters of methylene chloride. The mycelium is separated by fil tration and washed with about twice its volume of methylene chloride. The aqueous broth collected as a filtrate in the separation of the mycelium is stirred for an additional hour with a fresh portion of methylene chloride. The combined methylene chloride extracts, amounting to a total volume of about liters, are concentrated under reduced pressure to a volume of about 650 milliliters. This concentrate is washed with dilute sodium bicarbonate solution and with water and is then con centrated to dryness. The non-crystalline residue is dissolved in a 10 volume percent solution of ethyl acetate in benzene and fractionated on a silica gel chromatography column by elution of the column with mixtures of ethyl acetate and benzene containing gradually increasing percentages of ethyl acetate. In this manner there are obtained purified l5ot-hydroxy-4-pregnene-3,20- dione and 11,8,15a-dihydroxy-4-pregnene-3,20-dione, identical with the products of Example 1.

What is claimed is:

1. A process for the manufacture of a member of the group consisting of steroids oxygenated at position 11 and steroids oxygenated at position 15 which comprises: subjecting a steroid compound having a carbon skeleton of fewer than 22 carbon atoms and a methylene group in at least one of positions 11 and 15 to the oxygenating activity of a fungus of the genus Nigrospora, and isolating the resulting fermented steroid.

2. A process for the manufacture of a steroid oxygenated at position 15 which comprises: subjecting a steroid compound having a carbon skeleton of fewer than 22 carbon atoms and a methylene group in position 15 to the oxygenating activity of a fungus of the genus Nigrospora, and isolating the resulting fermented steroid.

3. A process for the manufacture of a steroid oxygenated at position 15 which comprises: subjecting a steroid compound containing the pregnane carbon skeleton and a methylene group in position 15 to the oxygenating activity of a fungus of the genus Nigrospora, and isolating the resulting fermented steroid.

4. A process which comprises: growing a fungus of the genus Nigrospora in an aqueous nutrient medium, dispersing progesterone in said medium, maintaining said dispersion under aerobic agitated conditions, and isolating a 15-hydroxy-4-pregnene-3,ZO-dione.

5. A process which comprises: growing a fungus of the genus Nigrospora in an aqueous nutrient medium, dispersing 17a-hydroxy-4-pregnene-3,20-dione in said medium, maintaining said dispersion under aerobic agitated conditions, and isolating a 15,17a-dihydroxy-4-pregnene- 3,20-dione.

6. A process which comprises: growing a fungus of the genus Nigrospora in an aqueous nutrient medium, dispersing a member of the class consisting of 2l-hydroxy-4-pregnene-3,20-dione and 21-acetoxy-4-pregnene- 3,20-dione in said medium, maintaining said dispersion under aerobic agitated conditions, and isolating a 15,21- dihydroxy-4-pregnene-3,ZO-dione.

7. A process for the manufacture of a steroid oxygenated at position 15 which comprises: aerobically contacting a steroid compound having a carbon skeleton of fewer than 22 carbon atoms and a methylene group in position 15 with a living culture of a fungus of the genus Nigrospora, and isolating the resulting fermented steroid.

8. A process for the manufacture of a steroid oxygenated at position 15 which comprises: aerobically contacting a steroid compound having a carbon skeleton of fewer than 22 carbon atoms and a methylene group in position 15 with a living culture of Nigrospora oryzae and isolating the resulting fermented steroid.

9. A process which comprises: growing an organism of the species Nigrospora oryzae in an aqueous nutrient medium, dispersing progesterone in said medium, maintaining said dispersion under aerobic agitated conditions, and isolating a member of the class consisting of a 15- hydroxy-4-pregnene-3,20-dione and an 11,15-dihydroxy- 4-pregnene-3,20-dione.

References Cited in the file of this patent Meister et al.: Jour. Am. Chem. Soc., 76, Aug. 5, 1954, pages 4050-4051.

Disclaimer 2,823,17O.Rooo1"t D. Muir, Deerfield, and Raymond M. Dodson, Park Ridge, I11. PROCESS FOR THE OXYGENATION OF STEROIDS WITH NIGROSPORA. Patent dated Feb. 11, 1958. Disclaimer filed Oct. 21, 1965, by the assignee G. D. Seewle c@ 00.

Hereby enters this disclaimer to claims 1 and 9 of said. patent.

[Ofioz'al Gazette April 5 1.966]

UNITED STATES PATENT OFFICE Certificate of Correction Patent No. 2,823,170 v February 11, 1958 Robert D. Muir et a1.

It is hereby certified that error appears in the printed specification of the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 2, line 10, for efiicint read eflicient; column 6, line 9, for -hyd0xyread -hydroxycolumn 7, line 21, for 173- read 17acolumn 10, line 10, for 11,8,15aread -l1a,1 5B- Signed and sealed this 29th day of April .1958.

[sEAL] Attest: J KARL H. AXLINE, RQBERT C. t A ".-Z

Attestz'ngOfiicer, Commissioner of Patents, 

1. A PROCESS FOR THE MANUFACTURE OF A MEMBER OF THE GROUP CONSISTING OF STERIODS OXYGENATED AT POSITION 11 AND STEROIDS OXYGENATED AT POSITION 15 WHICH COMPRISES: SUBJECTING A STEROID COMPOUND HAVING A CARBON SKELETON OF FEWER THAN 22 CARBON ATOMS AND A METHYLENE GROUP IN AT LEAST ONE OF POSITIONS 11 AND 15 TO THE OXYGENATING ACTIVITY OF A FUNGUS OF THE GENUS NIGROSPORA, AND ISOLATING THE RESULTING FERMENTED STEROID. 